An anti-inflammatory role for C/EBP? in human brain pericytes.
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ABSTRACT:
SUBMITTER: Rustenhoven J
PROVIDER: S-EPMC4499812 | biostudies-other | 2015
REPOSITORIES: biostudies-other
Publications
An anti-inflammatory role for C/EBPδ in human brain pericytes.
Rustenhoven Justin J Scotter Emma L EL Jansson Deidre D Kho Dan T DT Oldfield Robyn L RL Bergin Peter S PS Mee Edward W EW Faull Richard L M RL Curtis Maurice A MA Graham Scott E SE Park Thomas I-H TI Dragunow Mike M
Scientific reports 20150713
Neuroinflammation contributes to the pathogenesis of several neurological disorders and pericytes are implicated in brain inflammatory processes. Cellular inflammatory responses are orchestrated by transcription factors but information on transcriptional control in pericytes is lacking. Because the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) is induced in a number of inflammatory brain disorders, we sought to investigate its role in regulating pericyte immune responses. Ou ...[more]
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Project description:Brain inflammation plays a key role in neurological disease. Although much research has been conducted investigating inflammatory events in animal models, potential differences in human brain versus rodent models makes it imperative that we also study these phenomena in human cells and tissue.Primary human brain cell cultures were generated from biopsy tissue of patients undergoing surgery for drug-resistant epilepsy. Cells were treated with pro-inflammatory compounds IFN?, TNF?, IL-1?, and LPS, and chemokines IP-10 and MCP-1 were measured by immunocytochemistry, western blot, and qRT-PCR. Microarray analysis was also performed on late passage cultures treated with vehicle or IFN? and IL-1?.Early passage human brain cell cultures were a mixture of microglia, astrocytes, fibroblasts and pericytes. Later passage cultures contained proliferating fibroblasts and pericytes only. Under basal culture conditions all cell types showed cytoplasmic NF?B indicating that they were in a non-activated state. Expression of IP-10 and MCP-1 were significantly increased in response to pro-inflammatory stimuli. The two chemokines were expressed in mixed cultures as well as cultures of fibroblasts and pericytes only. The expression of IP-10 and MCP-1 were regulated at the mRNA and protein level, and both were secreted into cell culture media. NF?B nuclear translocation was also detected in response to pro-inflammatory cues (except IFN?) in all cell types. Microarray analysis of brain pericytes also revealed widespread changes in gene expression in response to the combination of IFN? and IL-1? treatment including interleukins, chemokines, cellular adhesion molecules and much more.Adult human brain cells are sensitive to cytokine challenge. As expected 'classical' brain immune cells, such as microglia and astrocytes, responded to cytokine challenge but of even more interest, brain pericytes also responded to such challenge with a rich repertoire of gene expression. Immune activation of brain pericytes may play an important role in communicating inflammatory signals to and within the brain interior and may also be involved in blood brain barrier (BBB) disruption . Targeting brain pericytes, as well as microglia and astrocytes, may provide novel opportunities for reducing brain inflammation and maintaining BBB function and brain homeostasis in human brain disease.
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Project description:BACKGROUND:Pericytes and endothelial cells are critical cellular components of the blood-brain barrier (BBB) and play an important role in neuroinflammation. To date, the majority of inflammation-related studies in endothelia and pericytes have been carried out using immortalised cell lines or non-human-derived cells. Whether these are representative of primary human cells is unclear and systematic comparisons of the inflammatory responses of primary human brain-derived pericytes and endothelia has yet to be performed. METHODS:To study the effects of neuroinflammation at the BBB, primary brain endothelial cells and pericytes were isolated from human biopsy tissue. Culture purity was examined using qPCR and immunocytochemistry. Electrical cell-substrate impedance sensing (ECIS) was used to determine the barrier properties of endothelial and pericyte cultures. Using immunocytochemistry, cytometric bead array, and ECIS, we compared the responses of endothelia and pericytes to a panel of inflammatory stimuli (IL-1β, TNFα, LPS, IFN-γ, TGF-β1, IL-6, and IL-4). Secretome analysis was performed to identify unique secretions of endothelia and pericytes in response to IL-1β. RESULTS:Endothelial cells were pure, moderately proliferative, retained the expression of BBB-related junctional proteins and transporters, and generated robust TEER. Both endothelia and pericytes have the same pattern of transcription factor activation in response to inflammatory stimuli but respond differently at the secretion level. Secretome analysis confirmed that endothelia and pericytes have overlapping but distinct secretome profiles in response to IL-1β. We identified several cell-type specific responses, including G-CSF and GM-CSF (endothelial-specific), and IGFBP2 and IGFBP3 (pericyte-specific). Finally, we demonstrated that direct addition of IL-1β, TNFα, LPS, and IL-4 contributed to the loss of endothelial barrier integrity in vitro. CONCLUSIONS:Here, we identify important cell-type differences in the inflammatory response of brain pericytes and endothelia and provide, for the first time, a comprehensive profile of the secretions of primary human brain endothelia and pericytes which has implications for understanding how inflammation affects the cerebrovasculature.
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Project description:Crosstalk mechanisms between pericytes, endothelial cells, and astrocytes preserve integrity and function of the blood-brain-barrier (BBB) under physiological conditions. Long intercellular channels allowing the transfer of small molecules and organelles between distant cells called tunneling nanotubes (TNT) represent a potential substrate for energy and matter exchanges between the tripartite cellular compartments of the BBB. However, the role of TNT across BBB cells under physiological conditions and in the course of BBB dysfunction is unknown. In this work, we analyzed the TNT's role in the functional dialog between human brain endothelial cells, and brain pericytes co-cultured with human astrocytes under normal conditions or after exposure to ischemia/reperfusion, a condition in which BBB breakdown occurs, and pericytes participate in the BBB repair. Using live time-lapse fluorescence microscopy and laser-scanning confocal microscopy, we found that astrocytes form long TNT with pericytes and endothelial cells and receive functional mitochondria from both cell types through this mechanism. The mitochondrial transfer also occurred in multicellular assembloids of human BBB that reproduce the three-dimensional architecture of the BBB. Under conditions of ischemia/reperfusion, TNT formation is upregulated, and astrocytes exposed to oxygen-glucose deprivation were rescued from apoptosis by healthy pericytes through TNT-mediated transfer of functional mitochondria, an effect that was virtually abolished in the presence of TNT-destroying drugs. The results establish a functional role of TNT in the crosstalk between BBB cells and demonstrate that TNT-mediated mitochondrial transfer from pericytes rescues astrocytes from ischemia/reperfusion-induced apoptosis. Our data confirm that the pericytes might play a pivotal role in preserving the structural and functional integrity of BBB under physiological conditions and participate in BBB repair in brain diseases.
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Project description:CCAAT enhancer-binding protein (C/EBP)? regulates gene expression in multiple organ systems and cell types, including astrocytes in the central nervous system (CNS). Inflammatory stimuli, interleukin (IL)-1?, tumor necrosis factor-?, human immunodeficiency virus (HIV)-1 and lipopolysaccharide induce astrocyte C/EBP? expression. C/EBP? is detectable in brains of Alzheimer's disease (AD), Parkinson's disease (PD) and HIV-1-associated dementia (HAD) patients, yet little is known about how C/EBP? contributes to astrocyte gene regulation during neuroinflammation.The expression of 92 human inflammation genes was compared between IL-1?-treated primary human astrocytes and astrocytes transfected with C/EBP?-specific small interfering (si)RNA prior to IL-1? treatment for 12 h. Transcripts altered by>two-fold compared to control were subjected to one-way analysis of variance and Newman-Keuls post-test for multiple comparisons. Expression of two genes, cyclooxygenase-2 (COX-2) and bradykinin receptor B2 (BDKRB2) was further confirmed in additional human astrocyte donors. Astrocytes were treated with mitogen-activated protein kinase-selective inhibitors, then with IL-1? for 12 or 24 h followed by COX-2 and BDKRB2, expression analyses.IL-1? altered expression of 29 of 92 human inflammation genes by at least two-fold in primary human astrocytes in 12 h. C/EBP? knockdown affected expression of 17 out of 29 IL-1?-regulated genes by>25%. Two genes relevant to neuroinflammation, COX-2 and BDKRB2, were robustly decreased and increased, respectively, in response to C/EBP? knockdown, and expression was confirmed in two additional donors. COX-2 and BDKRB2 mRNA remained altered in siRNA-transfected astrocytes at 12, 24 or 72 h. Inhibiting p38 kinase (p38K) activation blocked IL-1?-induced astrocyte COX-2 mRNA and protein expression, but not IL-1?-induced astrocyte BDKRB2 expression. Inhibiting extracellular-regulated kinase (ERK)1/2 activation blocked IL-1?-induced BDKRB2 mRNA expression while increasing COX-2 expression.These data support an essential role for IL-1? in the CNS and identify new C/EBP? functions in astrocytes. Additionally, this work suggests p38K and ERK1/2 pathways may regulate gene expression in a complementary manner to fine tune the IL-1?-mediated astrocyte inflammatory response. Delineating a role for C/EBP? and other involved transcription factors in human astrocyte inflammatory response may lead to effective therapies for AD, PD, HAD and other neurological disorders.
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